Peroxisome Proliferator-Activated Receptor Ligand

ABSTRACT

An object of the present invention is to provide a peroxisome proliferator-activated receptor γ (PPARγ) ligand derived from a natural product and to provide a composition for prevention or improvement of insulin resistant syndrome, diabetes mellitus, obesity, or visceral fat obesity, characterized by comprising the ligand as an active ingredient. The present invention provides a PPARγ ligand comprising as an active ingredient, at least one compound selected from the group consisting of coumaperine and derivatives thereof. The present invention also provides a composition for prevention or improvement of insulin resistant syndrome, diabetes mellitus, obesity, or visceral fat obesity comprising the compound as an active ingredient.

TECHNICAL FIELD

The present invention relates to a peroxisome proliferator-activatedreceptor γ ligand and to a composition for prevention or improvement ofvisceral fat obesity and related conditions and syndromes thereof.

BACKGROUND ART

Peroxisome proliferator-activated receptors (PPARs) are ligand-dependenttranscriptional regulators belonging to a nuclear receptor family, whichwere identified as transcriptional regulators to control the expressionof a gene cluster that maintains lipid metabolism. Three subtypes,PPARα, PPARδ (PPARβ, NUC-1, FAAR), and PPARγ, are known in mammals.PPARα is expressed mainly in the liver, and PPARδ is expresseduniversally. PPARγ includes two isoforms, PPARγ1 and PPARγ2. PPARγ1 isexpressed in adipose tissues as well as in immune-system organs, adrenalglands, and small intestine. PPARγ2 is expressed specifically in adiposetissues and is a master regulator that regulates the differentiation andmaturation of adipocytes (Non-Patent Document 1: T. Kawada, Progress inMedicine (Igaku no Ayumi in Japanese), 184, 519-523, 1998).

Known PPARγ ligands include: arachidonic acid metabolites such as15-deoxy-Δ12,14-prostaglandin J2 and Δ12-prostaglandin J2; unsaturatedfatty acids such as ω-3 polyunsaturated fatty acid, α-linolenic acid,eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA); andeicosanoids such as 9-hydroxyoctadecadienoic acid and13-hydroxyoctadecadienoic acid (Non-Patent Document 2: J. Auwerx,Diabetologia, 42, 1033-1049, 1999). Moreover, it has been disclosed thatconjugated unsaturated fatty acids having conjugated triene structuresor conjugated tetraene structures and having 10 to 26 carbon atoms arealso PPARγ ligands (Patent Document 1: Japanese Patent Laid-Open No.2000-355538). Furthermore, among synthetic compounds, thiazolidinederivatives such as troglitazone, pioglitazone, and rosiglitazone havebeen known to be PPARγ ligands.

The thiazolidine derivatives serving as PPARγligands were developed asinsulin resistance-improving drugs for type II diabetes mellitus(non-insulin-dependent diabetes mellitus: NIDDM), since the link of thethiazolidine derivatives to insulin resistance-improving effectsreceived attention because of the correlation of their agonistactivities with hypoglycemic effects. Specifically, the thiazolidinederivatives serving as PPARγ ligands activate PPARγ and thereby improveinsulin resistance by causing an increase in the number of normallyfunctioning small adipocytes differentiated from precursor adipocytes aswell as an apoptosis-induced decrease in the number of enlargedadipocytes, wherein the production and secretion of TNF-α or free fattyacid has been enhanced (Non-Patent Document 3: A. Okuno, et al., Journalof Clinical Investigation, 101, 1354-1361, 1998). The PPARγ ligandsimprove insulin resistance, and therefore, are also effective for theprevention or improvement of not only type II diabetes mellitus but alsoinsulin resistant syndrome such as hyperinsulinemia, lipid metabolismabnormality, obesity, hypertension, and arteriosclerotic diseases(Non-Patent Document 4: R. A. Degronze, et al., Diabetes Care, 14,173-194, 1991). It has been reported about their effects on obesity thatthe administration of troglitazone to patients with type II diabetesmellitus reduces visceral fat (Non-Patent Document 5: I. E. Kelly, etal., Diabetes Care, 22, 288-293, 1999; and Non-Patent Document 6: Y.Mori, et al., Diabetes Care, 22, 908-912, 1999). Therefore, the PPARγligands are also effective for the prevention or improvement of visceralfat obesity.

Coumaperine and derivatives thereof are components contained in naturalplants such as spices typified by Piper nigrum L. Coumaperine has shownto have physiological functions such as antioxidative effects(Non-Patent Document 7: Environmental Health Perspectives 67, 135-142,1986) and inhibitory effects on carcinogenesis (Patent Document 2:Japanese Patent Laid-Open No. 11-12174).

Patent Document 1: Japanese Patent Laid-Open No. 2000-355538 PatentDocument 2: Japanese Patent Laid-Open No. 11-12174 Non-Patent Document1: T. Kawada, Progress in Medicine (Igaku no Ayumi in Japanese), 184,519-523, 1998 Non-Patent Document 2: J. Auwerx, Diabetologia 42,1033-1049, 1999 Non-Patent Document 3: A. Okuno, et al., Journal ofClinical Investigation, 101, 1354-1361, 1998 Non-Patent Document 4: R.A. Degronze, et al., Diabetes Care, 14, 173-194, 1991 Non-PatentDocument 5: I. E. Kelly, et al., Diabetes Care, 22, 288-293, 1999Non-Patent Document 6: Y. Mori, et al., Diabetes Care, 22, 908-912, 1999Non-Patent Document 7: Environmental Health Perspectives 67, 135-142,1986 DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

In view of the foregoing, an object of the present invention is toprovide a PPARγ ligand and to provide a composition capable of treatingor preventing visceral fat obesity or type II diabetes mellitus andfurther capable of treating or preventing insulin resistant syndrome,metabolic syndrome, or visceral fat syndrome.

Means for Solving the Problems

The present inventors have found for the first time that coumaperine andderivatives thereof have PPARγ ligand activities and have consequentlycompleted the present invention. Specifically, the present inventionprovides the following inventions:

(1) A peroxisome proliferator-activated receptor γ ligand comprising asan active ingredient, at least one compound selected from the groupconsisting of coumaperine and derivatives thereof.

(2) The peroxisome proliferator-activated receptorγ ligand according to(1), wherein the compound is at least one compound selected from thegroup consisting of N-5-(4-hydroxyphenyl)-2E,4E-pentadienoyl piperidine,N-trans-feruloyl tyramine, N-trans-feruloyl piperidine,N-5-(4-hydroxy-3-methoxyphenyl)-2E,4E-pentadienoyl piperidine, andN-5-(4-hydroxy-3-methoxyphenyl)-2E-pentenoyl piperidine, salts thereof,and esterified forms thereof.

(3) A composition for prevention or treatment of visceral fat obesitycomprising as an active ingredient, at least one compound selected fromthe group consisting of coumaperine and derivatives thereof. (4) Acomposition for prevention or treatment of type II diabetes mellituscomprising as an active ingredient, at least one compound selected fromthe group consisting of coumaperine and derivatives thereof. (5) Acomposition for prevention or treatment of insulin resistant syndromecomprising as an active ingredient, at least one compound selected fromthe group consisting of coumaperine and derivatives thereof. (6) Acomposition for prevention or treatment of metabolic syndrome comprisingas an active ingredient, at least one compound selected from the groupconsisting of coumaperine and derivatives thereof. (7) A composition forprevention or treatment of visceral fat syndrome comprising as an activeingredient, at least one compound selected from the group consisting ofcoumaperine and derivatives thereof.

(8) The composition according to any of (3) to (7), wherein the compoundis one or more compound(s) selected from the group consisting ofN-5-(4-hydroxyphenyl)-2E,4E-pentadienoyl piperidine, N-trans-feruloyltyramine, N-trans-feruloyl piperidine,N-5-(4-hydroxy-3-methoxyphenyl)-2E,4E-pentadienoyl piperidine, andN-5-(4-hydroxy-3-methoxyphenyl)-2E-pentenoyl piperidine, salts thereof,and esterified forms thereof.

(9) The composition according to any of (3) to (7), wherein thecomposition comprises 0.1% by weight to 99% by weight in total of thecompound(s) selected from the group consisting of coumaperine andderivatives thereof. ADVANTAGES OF THE INVENTION

The present invention provides a peroxisome proliferator-activatedreceptor γ (PPARγ) ligand. A composition of the present invention iscapable of treating or preventing visceral fat obesity or type IIdiabetes mellitus and further capable of treating or preventing insulinresistant syndrome, metabolic syndrome, or visceral fat syndrome.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the embodiments of the present invention will be describedin detail.

Peroxisome proliferator-activated receptors (PPARs) used herein areligand-dependent transcriptional regulators belonging to a nuclearreceptor family, which were identified as transcriptional regulators tocontrol the expression of a gene cluster that maintains lipidmetabolism. PPARγ, one of subtypes thereof, is encoded by chromosome3p25 in the human genome (PPARα and PPARδ are encoded by chromosomes22q12-q13.1 and 6p21.2-p21.1, respectively) (Folia Pharmacol. Jpn., 117,319-327, 2001).

A PPARγ ligand of the present invention comprises as an activeingredient, at least one compound selected from the group consisting ofcoumaperine and derivatives thereof. The ligand used herein is anagonist or antagonist. The ligand of the present invention is preferablyan agonist from the viewpoint of treating or preventing visceral fatobesity and so on. Whether a compound has PPARγ ligand activities can beconfirmed by, for example, an assay described in Example 2 below.

The coumaperine used herein is N-5-(4-hydroxyphenyl)-2E,4E-pentadienoylpiperidine (I), which is a Piper nigrum L. spice-derived compoundrepresented by the formula (I) below.

Examples of the coumaperine derivatives of the present inventioninclude, but not particularly limited to, phenolic amide compounds suchas N-trans-feruloyl tyramine (II), N-trans-feruloyl piperidine (III),N-5-(4-hydroxy-3-methoxyphenyl)-2E,4E-pentadienoyl piperidine (IV), andN-5-(4-hydroxy-3-methoxyphenyl)-2E-pentenoyl piperidine (V) representedby the formulas (II) to (V) below. Further examples thereof includesalts, oxidized forms, reduced forms, glycosides, esterified forms,acetylated forms, and methylated forms of these compounds. Thosecompounds may be plant-derived or chemically synthesized.

In the present invention, the coumaperine or derivatives thereof arepreferably N-5-(4-hydroxyphenyl)-2E,4E-pentadienoyl piperidine (I)(coumaperine), N-trans-feruloyl tyramine (II), N-trans-feruloylpiperidine (III), N-5-(4-hydroxy-3-methoxyphenyl)-2E,4E-pentadienoylpiperidine (IV), N-5-(4-hydroxy-3-methoxyphenyl)-2E-pentenoyl piperidine(V), or salts or esterified forms thereof, more preferablyN-5-(4-hydroxyphenyl)-2E,4E-pentadienoyl piperidine (I) (coumaperine),N-trans-feruloyl tyramine (II), N-trans-feruloyl piperidine (III),N-5-(4-hydroxy-3-methoxyphenyl)-2E,4E-pentadienoyl piperidine (IV), orN-5-(4-hydroxy-3-methoxyphenyl)-2E-pentenoyl piperidine (V). Of course,they may be used alone or in combination of two or more of them.

The coumaperine and derivatives thereof used herein are found in naturalproducts and, particularly preferably, can be separated and collectedfrom Piper nigrum L. Alternatively, they can be separated and collectedfrom Piper nigrum L. oleoresin extracted from powdered dry fruits ofPiper nigrum L. with an organic solvent or the like.

In the present invention, a method for obtaining the coumaperine andderivatives thereof from Piper nigrum L. or Piper nigrum L. oleoresin isnot particularly limited. For example, powdered dry fruits of Pipernigrum L. or Piper nigrum L. oleoresin are treated with an organicsolvent, and insoluble components are separated to obtain an organicsolvent layer. Examples of the organic solvent utilized in thistreatment include n-hexane, methylene chloride, and ethylene dichloride.

Subsequently, this organic solvent layer is treated with, for example,an aqueous bicarbonate solution. The obtained organic solvent layer isfurther treated with an aqueous alkali solution. This aqueous alkalisolution layer is adjusted from neutral to acidic pH. Examples of thebicarbonate utilized in the treatment include potassium hydrogencarbonate, sodium hydrogen carbonate, potassium carbonate, sodiumcarbonate, and combinations thereof. Examples of a pH adjuster includehydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, lacticacid, citric acid, and combinations thereof.

Subsequently, the prepared solution can be extracted with an organicsolvent to thereby obtain a separated product containing several speciesof phenolic amid compounds. The obtained phenolic amide compounds can beeluted, for example, with a mixed solvent of methylene chloride andmethanol through a silica gel column to isolate the coumaperine andderivatives thereof (I) to (V).

In addition, the coumaperine and derivatives thereof (I) to (V) can alsobe obtained by a method described in Environmental Health Perspectives,67, 135-142, 1986.

Furthermore, the coumaperine and derivatives thereof of the presentinvention can also be obtained by synthesis. The coumaperine andderivatives thereof (I) to (V) can be synthesized by, but not limitedto, the methods described in, for example, Agricultural and BiologicalChemistry, 44, 2831, 1980, Tetrahedron, 59, 5337, 2003, and JapanesePatent Publication No. 1-21951.

Specifically, for example, bromocrotonic acid dissolved in dry benzeneat 0° C. is added to thionyl bromide in dry benzene. The solvent isdistilled off. The obtained bromide of 4-bromocrotonic acid is dissolvedin dry benzene and reacted with piperidine at approximately 0° C. Thereaction solution is heated to room temperature, then poured to chilledwater for additional 1 hour, and extracted with benzene. The organicsolvent layer is washed with an aqueous saturated NaHCO₃ solution andwater. After drying and concentration, syrup is obtained. This syrup ispurified by silica gel column chromatography to obtain an amide.

The purified amide is added to triethylphosphite, for example, at 100 to110° C. The reaction temperature is raised and kept for an appropriatetime. Excessive triethylphosphite is distilled off under reducedpressure. The residue is dissolved in dry DMF and mixed with a dry DMFsolution of p-benzyloxybenzaldehyde. This mixed solution is treated withan NaOEt solution and stirred at room temperature. The mixed solution isdiluted with water and extracted with methylene chloride. The driedextract is recrystallized from benzene to obtain white needles.

Concentrated hydrochloric acid is added to the acetic acid solution ofbenzylamide thus obtained. The mixed solution is treated atapproximately 100° C. and left at room temperature for an appropriatetime. The mixed solution is concentrated under vacuum, then supplementedwith water, and extracted with methylene chloride. The organic layer iswashed with an aqueous saturated NaHCO₃ solution to remove acetic acid.After extraction with a sodium hydroxide solution, the alkali extractthereof is made acidified with dilute hydrochloric acid. The aqueoussolution is extracted with methylene chloride. The concentrated productcan be recrystallized from acetone to obtain coumaperine needles.

The salts of the coumaperine and derivatives thereof (I) to (V) areintended to include the forms of non-toxic acid or base addition saltsthat can be formed from the compounds. For example, the compounds can betreated with an appropriate acid and thereby converted topharmaceutically acceptable acid addition salts thereof. In this case,exemplary acids include: inorganic acids such as hydrogen chloride,hydrogen bromide, hydrogen iodide, sulfuric acid, and phosphoric acid;and organic acids such as acetic acid, propionic acid, hydroxyaceticacid, lactic acid, pyruvic acid, glycolic acid, maleic acid, malonicacid, oxalic acid, benzenesulfonic acid, toluenesulfonic acid,methanesulfonic acid, trifluoroacetic acid, fumaric acid, succinic acid,malic acid, tartaric acid, citric acid, salicylic acid, p-aminosalicylicacid, pamoic acid, benzoic acid, and ascorbic acid. Examples of basesfor the base addition salt forms include: sodium, potassium, andcalcium; pharmaceutically acceptable amines such as ammonia, alkylamine,and benzathine; and amino acids such as arginine and lysine. The term“addition salts” used herein also includes solvates that can be formedfrom the compounds and salts thereof, for example, hydrates andalcoholates.

The oxidized forms, reduced forms, glycosides, esterified forms,acetylated forms, and methylated forms of the coumaperine andderivatives thereof (I) to (V) can respectively be obtained by treatingthe compounds with methods known in the art or can be obtained byextraction from plants containing them.

In the present invention, at least one compound selected from thecoumaperine and derivatives thereof that can be used is, but not limitedto, a pure compound. Semi-purified or crude products can also be used aslong as they do not contain impurities inappropriate as drugs or foods.

A “composition for treatment or prevention of visceral fat obesity ortype II diabetes mellitus” and a “composition for treatment orprevention of insulin resistant syndrome, metabolic syndrome, orvisceral fat syndrome” used herein are characterized by comprising as anactive ingredient, at least one compound selected from the groupconsisting of coumaperine and derivatives thereof.

The insulin resistant syndrome used herein means a disease group that ischaracterized by the presence of two conditions, insulin resistance andhyperinsulinemia, and complicates one or more related lesion(s) ofobesity, type II diabetes mellitus, hypertension, arterioscleroticdiseases, or lipid metabolism abnormality (Netherlands Journal ofMedicine, 50, 191-197, 1997).

The metabolic syndrome used herein is a syndrome corresponding to a casethat has abdominal obesity (particularly, visceral fat obesity) as abasic condition and additionally has multiple risk factors such asfasting hyperglycemia, hypertriglyceridemia, hypo-HDL-cholesterolemia,and hypertension (Circulation Journal, 68, 975-981, 2004).

Diagnostic criteria of metabolic syndrome differ from nation to nationand, in Japan, are as follows (Internal Medicine, 94, 188-202, 2005):

-   -   Abdominal obesity: abdominal circumference for male≧85 cm, for        female≧90 cm Besides, two or more items of the followings:    -   Hypertriglyceridemia≧150 mg/dl and/or        Hypo-HDL-cholesterolemia<40 mg/dL    -   Fasting hyperglycemia≧110 mg/dL    -   Systolic blood pressure≧130 mmHg and/or Diastolic blood        pressure≧85 mmHg

The visceral fat syndrome used herein is a disease group thatcomplicates five conditions, visceral fat accumulation, abnormal glucosetolerance, hyperlipidemia, hypertension, and hypo-HDL-cholesterolemia(Internal Medicine, 81, 1831-1835, 1992).

Visceral fat obesity is common in men and is different from subcutaneousfat obesity common in women. In studies on the development andprogression of risk factors causing arteriosclerosis, insulin resistanceis placed in a rank higher than risk factors such as hyperlipidemia,diabetes mellitus, and hypertension, and visceral fat obesity is placedin the highest rank. Diagnostic criteria of obesity also placeimportance particularly on visceral fat as bad fat causing the onset ofthe syndrome, and the accumulation of visceral fat is said to increasethe risk (Circulation Journal, 66, 987-992, 2002).

PPARγ agonists are characterized by specifically reducing visceral fat.Therefore, the composition of the present invention can particularlytreat or prevent visceral fat obesity and thereby treats or preventsinsulin resistance, further, diabetes mellitus, hyperlipidemia,hypertension, and so on.

The content (in terms of the total weight) of the at least one compoundselected from the coumaperine and derivatives thereof in the compositionis not limited as long as it is suitable to the treatment or preventionof the diseases described above. The content can be, for example, 0.1 to100% by weight. The composition comprises 0.1 to 99% by weight, morepreferably 1% to 99% by weight of the compound(s), from the viewpoint ofsufficient effects. More preferably, the content can be 10 to 90% byweight.

The composition is not limited by form and can be used as, for example,food and drink such as food with health claims (food for specifiedhealth use and food with nutrient function claims) and health food, adrug, and a quasi drug.

The composition, when used as food and drink, can be ingested directlyor can be ingested after being prepared into a easily taken form such asa capsule, tablet, or granule by use of a carrier, auxiliary agent, orthe like known in the art.

Furthermore, the composition can be mixed with materials for food anddrink and thereby used in all foods and drinks including: confectionerysuch as chewing gums, chocolates, candies, jellies, biscuits, andcrackers; frozen desserts such as ice creams and ice cubes; drinks suchas tea, soft drinks, nutritional supplement drinks, and beautysupplement drinks; noodles such as udon noodle, Chinese noodle,spaghetti, and instant noodle; fish paste products such as boiled fishpaste (kamaboko), tube-shaped fish paste cake (chikuwa), and poundedfish cake (hanpen); seasonings such as dressing, mayonnaise, and sauce;oils and fats such as margarine, butter, and salad oil; and bread, ham,soup, pouch-packed foods, and frozen foods. These compositions for foodand drink may be ingested by one adult at a dose of usually 0.1 to 3000mg/kg of body weight, preferably 1 to 300 mg/kg of body weight, per dayin terms of the amount of the coumaperine or a derivative thereof. Thecomposition of the present invention can also be used as feed or petfood for livestock and pets and may be ingested at a dose of preferably0.1 to 3000 mg/kg of body weight per day in terms of the amount of thecoumaperine or a derivative thereof.

The composition, when used as a drug, is not limited by dosage form.Examples thereof include preparations such as capsules, tablets,granules, injections, suppositories, and patches. To make thecomposition into such preparations, other pharmaceutically acceptableformulation materials, for example, excipients, disintegrants,lubricants, binders, antioxidants, coloring agents, anti-aggregationagents, absorption promoters, solubilizers, and stabilizers can be addedappropriately thereto. These preparations are administered to one adultat a dose of usually 0.1 to 3000 mg/kg of body weight, preferably 1 to300 mg/kg of body weight, in a single or divided dose per day in termsof the amount of the coumaperine or a derivative thereof. Thecomposition can also be used as a drug for livestock and pets and isadministered at a dose of preferably 0.1 to 3000 mg/kg of body weightper day in terms of the amount of the coumaperine or a derivativethereof.

EXAMPLES

Hereinafter, the present invention will be described more specificallywith reference to Examples. However, the present invention is notintended to be limited to these Examples.

Example 1 Synthesis of Coumaperine

Coumaperine was prepared by a synthesis method described below.

Bromocrotonic acid (74.3 g, 0.03 M) dissolved in dry benzene (180 ml) at0° C. was added to thionyl bromide (34.8 ml, 0.01 M) in dry benzene (45ml). The solvent was distilled off. The obtained bromide of4-bromocrotonic acid was dissolved in dry benzene (75 ml) and reactedwith piperidine (89 ml, 0.08 M) at 0° C. The reaction solution washeated to room temperature, then poured to chilled water for additional1 hour, and extracted with benzene. The organic solvent layer was washedwith an aqueous saturated NaHCO₃ solution and water. After drying andconcentration, 32.4 g of syrup was obtained. This syrup was purified bysilica gel column chromatography to obtain an amide.

The purified amide was added to triethylphosphite (9.6 g) at 105° C. Thereaction temperature was raised to 150° C. and kept for 1 hour.Excessive triethylphosphite was distilled off under reduced pressure.The residue was dissolved in dry DMF (30 ml) and mixed with a dry DMFsolution (45 ml) containing 12.2 g of p-benzyloxybenzaldehyde. Thismixed solution was treated with an NaOEt solution and stirred at roomtemperature. The mixed solution was diluted with water and extractedwith methylene chloride. The dried extract (19.2 g) was recrystallizedfrom benzene to obtain white needles.

Concentrated hydrochloric acid (150 ml) was added to the acetic acidsolution (300 ml) of benzylamide (6 g) thus obtained. The mixed solutionwas treated at 100° C. for 2 hours and left overnight at roomtemperature. The mixed solution was concentrated under vacuum, thenmixed with water, and extracted with methylene chloride. The organiclayer was washed with an aqueous saturated NaHCO₃ solution to removeacetic acid. After extraction with 1N sodium hydroxide solution, thealkali extract thereof was made acidic with dilute hydrochloric acid.The aqueous solution was extracted with methylene chloride. Theconcentrated product (3 g) could be recrystallized from acetone toobtain coumaperine needles (2.1 g).

Example 2 PPARγ Ligand Activity

CV-1 cells (cultured cells derived from male African green monkeykidney) were seeded at 6×10³ cells/well into a 96-well culture plate andcultured at 37° C. for 24 hours under 5% CO₂ conditions. The medium usedwas DMEM (Dulbecco's Modified Eagle Medium; GIBCO) containing 10% FBS(fetal bovine serum), 10 ml/L penicillin/streptomycin solution (5000IU/ml and 5000 μg/ml, respectively; GIBCO), and 37 mg/L ascorbic acid(Wako Pure Chemical Industries, Ltd.). The cells were washed withOPTI-MEM (GIBCO) and then transfected with pM-mPPARγ and 4×UASg-luc byuse of Lipofectamine plus (GIBCO). The pM-mPPARγ is a chimeric proteinexpression plasmid in which a yeast-derived transcription factor GAL4gene (amino acid sequence at 1 to 147 positions) is ligated with a mousePPARγ ligand-binding site gene (amino acid sequence at 174 to 475positions). The 4×UASg-luc is a reporter plasmid in which four copies ofthe upstream activating sequence of GAL4 (UASg) are incorporatedupstream of a luciferase gene. Approximately 24 hours aftertransfection, the medium was replaced with a sample-containing medium(n=4), followed by culture for additional 24 hours. The sample dissolvedin dimethyl sulfoxide (DMSO) or DMSO used as an untreated control wasadded at 1/1000 of the volume to the medium. The cells were washed withCa- and Mg-containing phosphate-buffered saline (PBS+). Then, Luclite(Packard) was added thereto, and the luminescence intensity ofluciferase was measured with a TopCount microplatescintillation/luminescence counter (Packard).

pM (plasmid from which PPARγ ligand-binding site gene was removed) wasused as a control group instead of the pM-mPPARγ and subjected tomeasurement in the same way as for the measurement group. The ratio(measurement group/control group) of the average luminescence intensityvalues (n=4) between the measurement and control groups was calculatedfor each sample. Specific activity relative to the untreated control wasused as the PPARγ ligand activity of the sample. The results are shownin Table 1.

TABLE 1 Concentration PPARγ ligand added activity Untreated control(DMSO) (0.1%) 1.00 Troglitazone 0.5 μM 2.20 2 μM 4.05 10 μM 7.52Coumaperine 0.5 μg/ml 1.05 1 μg/ml 1.07 2 μg/ml 1.24 5 μg/ml 1.78 10μg/ml 2.07 20 μg/ml 2.53 30 μg/ml 2.38 50 μg/ml 2.44

Troglitazone (Sankyo Co., Ltd.) was used as a positive control tocompare the PPARγ ligand activity among the compounds. As seen fromTable 1, the coumaperine was observed to have a concentration-dependentPPARγ ligand activity.

Example 3 Toxicity Test on Coumaperine

A toxicity test was conducted on the coumaperine of the presentinvention.

Coumaperine was orally administered at 2000 mg/kg of body weight torats. As a result, no death case was observed, and toxic conditions werenot particularly observed therein. Thus, the coumaperine was confirmedto have no or exceedingly low toxicity.

Example 4 Preparation of Tablet Containing Coumaperine

Coumaperine 45 parts by weight Lactose 35 parts by weight Crystallinecellulose 15 parts by weight Sucrose fatty acid ester  5 parts by weight

A tablet for food and drink containing coumaperine was prepared by astandard method from the composition. Example 5 Preparation of SoftCapsule Containing Coumaperine

Coumaperine 40 parts by weight Sesame oil 55 parts by weight Glycerinfatty acid ester  5 parts by weight

A soft capsule for food and drink containing coumaperine was prepared bya standard method from the composition. Example 6 Preparation of CrackerContaining Coumaperine

Coumaperine 1 part by weight Soft flour 120 parts by weight Common salt1 part by weight Baking powder 2 parts by weight Butter 30 parts byweight Water 40 parts by weight

A cracker containing coumaperine was prepared by a standard method fromthe composition. Example 7 Preparation of Udon Noodle ContainingCoumaperine

Coumaperine 1 part by weight Hard flour 100 parts by weight Soft flour100 parts by weight Common salt 10 parts by weight Water 100 parts byweight

An udon noodle containing coumaperine was prepared by a standard methodfrom the composition. Example 8 Preparation of Dressing ContainingCoumaperine

Coumaperine 10 parts by weight Olive oil 80 parts by weight Vinegar 60parts by weight Common salt 3 parts by weight Pepper 1 part by weightLemon juice 5 parts by weightA dressing containing coumaperine was prepared by a standard method fromthe composition.

1. A peroxisome proliferator-activated receptor γ ligand comprising asan active ingredient, at least one compound selected from the groupconsisting of coumaperine and derivatives thereof.
 2. The peroxisomeproliferator-activated receptor γ ligand according to claim 1, whereinthe compound is at least one compound selected from the group consistingof N-5-(4-hydroxyphenyl)-2E,4E-pentadienoyl piperidine, N-trans-feruloyltyramine, N-trans-feruloyl piperidine,N-5-(4-hydroxy-3-methoxyphenyl)-2E,4E-pentadienoyl piperidine, andN-5-(4-hydroxy-3-methoxyphenyl)-2E-pentenoyl piperidine, salts thereof,and esterified forms thereof.
 3. A composition for prevention ortreatment of visceral fat obesity comprising as an active ingredient, atleast one compound selected from the group consisting of coumaperine andderivatives thereof.
 4. A composition for prevention or treatment oftype II diabetes mellitus comprising as an active ingredient, at leastone compound selected from the group consisting of coumaperine andderivatives thereof.
 5. A composition for prevention or treatment ofinsulin resistant syndrome comprising as an active ingredient, at leastone compound selected from the group consisting of coumaperine andderivatives thereof.
 6. A composition for prevention or treatment ofmetabolic syndrome comprising as an active ingredient, at least onecompound selected from the group consisting of coumaperine andderivatives thereof.
 7. A composition for prevention or treatment ofvisceral fat syndrome comprising as an active ingredient, at least onecompound selected from the group consisting of coumaperine andderivatives thereof.
 8. The composition according to claim 3, whereinthe compound is one or more compound(s) selected from the groupconsisting of N-5-(4-hydroxyphenyl)-2E,4E-pentadienoyl piperidine,N-trans-feruloyl tyramine, N-trans-feruloyl piperidine,N-5-(4-hydroxy-3-methoxyphenyl)-2E,4E-pentadienoyl piperidine, andN-5-(4-hydroxy-3-methoxyphenyl)-2E-pentenoyl piperidine, salts thereof,and esterified forms thereof.
 9. The composition according to claim 3,wherein the composition comprises 0.1% by weight to 99% by weight intotal of the compound(s) selected from the group consisting ofcoumaperine and derivatives thereof.
 10. The composition according toclaim 4, wherein the compound is one or more compound(s) selected fromthe group consisting of N-5-(4-hydroxyphenyl)-2E,4E-pentadienoylpiperidine, N-trans-feruloyl tyramine, N-trans-feruloyl piperidine,N-5-(4-hydroxy-3-methoxyphenyl)-2E,4E-pentadienoyl piperidine, andN-5-(4-hydroxy-3-methoxyphenyl)-2E-pentenoyl piperidine, salts thereof,and esterified forms thereof.
 11. The composition according to claim 5,wherein the compound is one or more compound(s) selected from the groupconsisting of N-5-(4-hydroxyphenyl)-2E,4E-pentadienoyl piperidine,N-trans-feruloyl tyramine, N-trans-feruloyl piperidine,N-5-(4-hydroxy-3-methoxyphenyl)-2E,4E-pentadienoyl piperidine, andN-5-(4-hydroxy-3-methoxyphenyl)-2E-pentenoyl piperidine, salts thereof,and esterified forms thereof.
 12. The composition according to claim 6,wherein the compound is one or more compound(s) selected from the groupconsisting of N-5-(4-hydroxyphenyl)-2E,4E-pentadienoyl piperidine,N-trans-feruloyl tyramine, N-trans-feruloyl piperidine,N-5-(4-hydroxy-3-methoxyphenyl)-2E,4E-pentadienoyl piperidine, andN-5-(4-hydroxy-3-methoxyphenyl)-2E-pentenoyl piperidine, salts thereof,and esterified forms thereof.
 13. The composition according to claim 7,wherein the compound is one or more compound(s) selected from the groupconsisting of N-5-(4-hydroxyphenyl)-2E,4E-pentadienoyl piperidine,N-trans-feruloyl tyramine, N-trans-feruloyl piperidine,N-5-(4-hydroxy-3-methoxyphenyl)-2E,4E-pentadienoyl piperidine, andN-5-(4-hydroxy-3-methoxyphenyl)-2E-pentenoyl piperidine, salts thereof,and esterified forms thereof.
 14. The composition according to claim 4,wherein the composition comprises 0.1% by weight to 99% by weight intotal of the compound(s) selected from the group consisting ofcoumaperine and derivatives thereof.
 15. The composition according toclaim 5, wherein the composition comprises 0.1% by weight to 99% byweight in total of the compound(s) selected from the group consisting ofcoumaperine and derivatives thereof.
 16. The composition according toclaim 6, wherein the composition comprises 0.1% by weight to 99% byweight in total of the compound(s) selected from the group consisting ofcoumaperine and derivatives thereof.
 17. The composition according toclaim 7, wherein the composition comprises 0.1% by weight to 99% byweight in total of the compound(s) selected from the group consisting ofcoumaperine and derivatives thereof.